Cellular arrays comprising encoded cells

ABSTRACT

A biosensor, sensor array, sensing method and sensing apparatus are provided in which individual cells or randomly mixed populations of cells, having unique response characteristics to chemical and biological materials, are deployed in a plurality of discrete sites on a substrate. In a preferred embodiment, the discrete sites comprise microwells formed at the distal end of individual fibers within a fiber optic array. The biosensor array utilizes an optically interrogatable encoding scheme for determining the identity and location of each cell type in the array and provides for simultaneous measurements of large number of individual cell respnses to target analyses. The sensing method utilizes the unique ability of cell populations to respond to biologically significant compounds in a characteristic and detectable manner. The biosensor array and measurement method may be employed in the study of biologically active materials in situ environmental monitoring, monitoring of a variety of bioprocesses, and for high throughput screening of large combinatorial chemical libraries.

The present application claims the benefit of U.S. Application Ser. No.60/230,007, which is expressly incorporated herein by reference.

FIELD OF THE INVENTION

The present invention is directed to biosensors, biosensor arrays,sensing apparatus and sensing methods which employ cells and mixedpopulations of cells, particularly encoded cells, for analysis ofchemical and biological materials on cellular processes.

BACKGROUND OF THE INVENTION

It is generally recognized that important technical advances inchemistry, biology and medicine benefit from the ability to performmicroanalysis of samples in minute quantities. However, makinganalytical measurements on minute quantities has long been a challengedue to difficulties encountered with small volume sample handling,isolation of analyses, and micro-analysis of single-cell physiology.

Nanoliter, picoliter, and femtoliter volume studies have been exploredin a range of applications involving in vitro and in vivo cellularinvestigations [R. M. Wightman, et al., Proc. Natl. Acad. Sci. U.S.A.88: 10754 (1991); R. H. Chow, et al. Nature 356: 60 (1992); T. K. Chen,et al. Anal. Chem. 66: 3031 (1994); S. E. Zerby, et al., Neurochem. 66:651 (1996); P. A. Garis, et al. J. Neurosci. 14: 6084 (1994); G. Chen,et al., J. Neurosci. 15: 7747 (1995)], electrochemistry [R. A. Clark, etal., Anal. Chem. 69(2): 259 (1997)], matrix assisted laserdesorption-ionization mass spectrometry [S. Jespersen, et al., RapidCommun. Mass Spectrom. 8: 581 (1994)], micro-column liquidchromatography [I. A. Holland, et al., Anal. Chem. 67: 3275 (1995); M.D. Oates, et al., Anal. Chem. 62: 1573 (1990)], micro-titration [M.Gratzl. et al Anal. Chem. 65: 2085 (1993); C. Yi, et al., Anal. Chem.66: 1976 (1994)], and capillary electrophoresis [M. Jansson, et al., J.Chromatogr. 626: 310 (1992); P. Beyer Hietpas, et al. J. Liq.Chromatogr. 18: 3557 (1995)].

Clark, et al. [Anal. Chem. 69(2): 259 (1997)] has disclosed a method forfabricating picoliter microvials for electrochemical microanalysis usingconventional photolithographic masking and photoresist techniques totransfer mold polystyrene microvials on silicon wafer templates. Thesemicrovials typically exhibit non-uniformity in size and shape due to thedifficulty in controlling the re-etching of the molding surface and thetransfer molding process.

Park, et al. [Science 276: 1401 (1997)] has disclosed a modifiedlithographic method for producing arrays of nanometer-sized holes usingpolystyrene-polybutadiene, ordered, diblock copolymers as masks inreactive ion etching of silicon nitride. This multi-step method iscapable of producing arrays of picoliter-sized holes which are typically20 nanometers in diameter and 20 nanometers deep with a spacing of 40nanometers. Hole densities of up to 10¹¹ holes/cm² are disclosed. Therange of sizes and spacings of the holes produced by this method islimited by the size of the copolymer microdomains. Uniformity of holesize and spacing is difficult to maintain with this method due todifficulties in controlling the etching method employed to form theholes.

Deutsch, et al. [Cytometry 16: 214 (1994)] have disclosed a porouselectroplated nickel microarray comprised of micron-sized conical holesin blackened nickel plate. Hole sizes range from a 7 um upper diameterto a 3 um lower diameter with an 8 um depth. The array is used as a cellcarrier for trapping individual cells while studying the responses ofindividual cells to changes in their microenvironment. In U.S. Pat. No.4,772,540, Deutsch, et al., have also disclosed a method for making suchan array using a combined photoresist and electroplating technique.

Corning Costar Corp. (Actor, Ma) produces a commercial microwell arrayfor miniaturized assays under the trademark PixWell™. These arrays aremade from microformed glass plates and comprise 40 um diameter by 20 umdeep tapered wells with a well density of 4356 wells/cm².

Microwell arrays have particular utility in the study of living cells.In cell research, the measurement of responses of individual cells tochanges or manipulations in their local environment is desirable. Anymethod or device designed for such studies must provide for thecapability of maintaining cell viability, identifying the location ofindividual cells, and correlating response measurements with individualcells.

Due to the availability of viable fluorescent probes for intracellularstudies, fluorescence measurements of living cells have significantutility in the study of cell functions. Thus fluorescence opticalmeasurements are often utilized in cell studies where three genericmethods of cell measurement are available, comprising bulk measurementsof cell populations, dynamic measurements of cell populations orindividual cells, and static measurements of individual cells.

The characteristics of an entire cell population as a whole can bestudied with bulk measurements of sample volumes having a plurality ofcells. This method is preferred where cell populations are veryhomogeneous. A generally recognized limitation of this method is thepresence of background fluorescence which reduces the sensitivity ofmeasurements and the inability of distinguishing differences orheterogeneity within a cell population.

Flow cytometry methods are often employed to reduce problems withbackground fluorescence which are encountered in bulk cell populationmeasurements [M. R. Gauci, et al., Cytometry 25: 388 (1996); R. C.Boltz, et al., Cytometry 17: 128 (1994)]. In these methods, cellfluorescence emission is measured as cells are transported through anexcitation light beam by a laminar flowing fluid. Flow cytometry methodsmay be combined with static methods for preliminary sorting anddepositing of a small number of cells on a substrate for subsequentstatic cell measurements [U.S. Pat. No. 4,009,435 to Hogg, et al.; Kanz,et al., Cytometry 7: 491 (1986); Schildkraut, et al., J. HistochemCytochem 27; 289 (1979)].

Gauci, et al., disclose a method where cell size, shape and volume ismeasured by light scattering and fluorescent dyes are utilized todetermine protein content and total nucleic acid content of cells. Thismethod further provides for counting and sizing various cells at a rateof approximately 100 cells per second.

Flow cytometry techniques are generally limited to short duration,single measurements of individual cells. Repetitive measurements on thesame cell over time are not possible with this method since typicaldwell times of a cell in the excitation light beam are typically a fewmicroseconds. In addition, the low cumulative intensity from individualcell fluorescence emissions during such short measurement times reducesthe precision and limits the reliability of such measurements.

Regnier, et al., [Trends in Anal. Chem. 14(4): 177 (1995)] discloses aninvasive, electrophoretically mediated, microanalysis method for singlecell analysis. The method utilizes a tapered microinjector at theinjection end of a capillary electrophoresis column to pierce anindividual cell membrane and withdraw a sample of cytoplasm. The methodmeasures cell contents, one cell at a time. The method is generallylimited to the detection of easily oxidized species.

Hogan, et al., [Trends in Anal. Chem. 12(1): 4 (1993)] discloses amicrocolumn separation technique which may be utilized in combinationwith either a conventional gas chromatograph-mass spectrometer, microthin layer chromatography or high pressure liquid manipulation of smallcellular volumes. The sensitivity of the method is limited and mayrequire pre-selection of target compounds for detection.

Static methods are generally the preferred method for measurements onindividual cells. Measurement methods range from observing individualcells with a conventional optical microscope to employing laser scanningmicroscopes with computerized image analysis systems [see L. Hart, etal., Anal. QuanL Cyto/. Histol. 12: 127 (1990)]. Such methods typicallyrequire the attachment of individual cells to a substrate prior toactual measurements. Problems are typically encountered in attachingsingle cells or single layers of cells to substrates and in maintainingcells in a fixed location during analysis or manipulation of the cellmicroenvironment. Additionally, repetitive measurements on individualcells typically require physically indexing the location of individualcells and providing a mechanism for scanning each cell sequentially andreturning to indexed cell locations for repeated analysis of individualcells.

Huang, et al., [Trends in Anal. Chem., 14(4) 158 (1995)] discloses astatic electrochemical method and electrode for monitoring thebiochemical environment of single cells. The method requires fabricationand manual positioning of a microelectrode reference and workingelectrode within the cell. The method has been used to detect insulin,nitric oxide and glucose inside single cells or external to the cells.The method is generally limited to the study of redox reactions withincells.

Ince, et al. [J. Immunol. Methods 128: 227 (1990)] disclose a closedchamber device for the study of single cells under controlledenvironments. This method employs a micro-perfusion chamber which iscapable of creating extreme environmental conditions for cell studies.Individual cells are held in place by two glass coverslips as varioussolutions are passed through the chamber. One limitation of the methodis the difficulty in eliminating entrapped gas bubbles which cause ahigh degree of autofluorescence and thus reduces the sensitivity ofmeasurements due to background fluorescence.

In an attempt to overcome the limitations encountered with conventionalstatic methods, Deutsch, et al., [Cytometry 16: 214 (1994)] and Weinreband Deutsch, in U.S. Pat. Nos. 4,729,949, 5,272,081, 5,310,674, and5,506,141, have disclosed an apparatus and method for repetitive opticalmeasurements of individual cells within a cell population where thelocation of each cell is preserved during manipulation of the cellmicroenvironment.

A central feature of the apparatus disclosed by Deutsch, et al., is acell carrier, comprising a two dimensional array of apertures or trapswhich are conical-shaped in order to trap and hold individual cells byapplying suction. The cell carrier is typically fabricated by thecombined electroplating-photoresist method disclosed in U.S. Pat. No.4,772,540 to Deutsch, et al. The purpose of the cell carrier is toprovide a means for maintaining the cells in fixed array locations whilemanipulating the cell environment. Individual cells are urged into cellcarrier holes by suction and the wells are subsequently illuminated witha low-intensity beam of polarized light that reads back-emittedpolarization and intensity. Measurements are compared when two differentreagents are sequentially reacted with the cells. The method asdisclosed requires two separate cell carriers for both a baselinecontrol and analyte measurement.

The method and device of Deutsch, et al., have been employed bypathologists in diagnostic tests to determine the health and viabilityof cell samples taken from patients. The method and device have beenapplied to both cancer screening [Deutsch, et al., Cytometry 16: 214(1994), Cytometry 23: 159 (1996), and European J. Cancer 32A (10): 1758(1996)] and rheumatoid arthritis [Zurgil, et al., Isr. J. Med. Sci. 33:273 (1997)] in which fluorescence polarization measurements are used todifferentiate lymphocytes of malignant versus healthy cells based onchanges in the internal viscosity and structuredness of the cytoplasmicmatrix induced by exposure to tumor antigen and mitogens.

The method and device disclosed by Deutsch, et al., requires employmentof a scanning table driven by three stepping motors and a computercontrol system for mapping, indexing and locating individual cells inthe cell carrier. The use of such mechanical scanning methods introduceslimitations in reproducibility and accuracy of measurements due toconventional mechanical problems encountered with backlash andreproducible positioning of individual cell locations for repeatedmeasurements. In addition, mechanical scanning of the entire arrayprolongs the measurement time for each cell in the array.

The method disclosed by Deutsch, et al., is further limited by the useof fluorescence polarization measurements which have certain intrinsiclimitations due to the significant influence of various optical systemcomponents on polarization as the fluorescence emission response ispassed from the cell carrier to optical detectors. Birindelli, et al.[European J. Cancer 33(8): 1333 (1997)], has also identified limitationsin this method due to fluctuations in electropolarisation values whichrequire taking averages of at least three measurement scans for eachcondition so as to obtain reliable measurements. In addition, for cellstudies, polarization measurements are generally limited to cellresponses which produce sufficient changes in cytoplasm viscosity toproduce a detectable change in polarization. Since not all cellresponses are accompanied by detectable viscosity changes, the method isfurther limited to the cell activities which create such viscositychanges in the cytoplasm.

Zare. et al., [Science 267: 74 (1995); Biophotonics International,March-April, p 17 (1995)] discloses a biosensor system based on theresponse of living cells to complex biological materials fractionated bya microcolumn separation technique. Cells which were positioned on aglass cover slip were treated with a fluorescent probe and subsequentlyshown to be sensitive to a series of biological compounds includingacetylcholine, bradykinin, and adenosine triphosphate as well as changesin intracellular calcium levels.

Yeung, et al. [Ace. Chem. Res. 27: 409 (1994)] has reviewed a number ofmethods for single cell response studies and has observed a significantvariation and heterogeneity within cell populations based on analytemeasurements. For example, the reference discloses a capillaryelectrophoresis method for exposing cells to biologically reactivecompounds, extracting the intracellular fluid of individual cellsproduced in response to such compounds, and identifying analyses frommigration times in the capillary column. Other fluorescence-based assaysare also disclosed. Significant cell-to-cell variations andheterogeneity in individual cell responses within a cell population wereobserved which differences could provide a means for discriminatingbetween biological and chemical compounds in contact with individualcells.

McConnell, et al. [Science, 257: 1906 (1992)], disclose amicrophysiometer device known as the “Cytosensor” which uses a lightaddressable potentiometer sensor to measure the rate at which cellsacidify their environment. This sensor acts as miniaturized pH electrodefor monitoring cell responses which produce detectable changes in localpH. The disclosed device is limited to the measurement of protonexcretions from cells and thus is only capable of detecting acidic cellresponses to analyses.

U.S. Pat. No. 5,177,012 to Kim, et al., disclose a biosensor for thedetermination of glucose and fructose. The biosensor is produced bytreating whole cells with an organic solvent and immobilizing thetreated cells residue on a support to form a whole cell membrane whichis applied to a pH electrode.

U.S. Pat. No. 5,690,894 to Pinkel, et al., discloses a biosensor whichemploys biological “binding partners”, materials such as nucleic acids,antibodies, proteins, lectins and other materials derived from cells,tissues, natural or genetically-engineered organisms. These agents areused in conjunction with a fiber optic array where each species ofbinding partners is uniquely addressed by a group of fibers within thefiber optic bundle which is coupled to an optical detector. The arraywas designed for screening of extensive arrays of biological bindingpartners.

While many of the prior art methods provide for the analysis of eithersingle cells or populations of cells and some of these methods providefor monitoring cell responses to target analyses, none of the disclosedmethods provides for employing large populations of monocultures ormixed populations of living cells for simultaneously monitoring theresponses of individual cells to biological stimuli produced by chemicaland biological analyses. Thus there is a need for a biosensor array andmethod which efficiently utilizes the ability of populations of livingcells to respond to biologically significant compounds in a unique anddetectable manner. Since the selectivity of living cells for suchcompounds has considerable value and utility in drug screening andanalysis of complex biological fluids, a biosensor which makes use ofthe unique characteristics of living cell populations would offerdistinct advantages in high throughput screening of combinatoriallibraries where hundreds of thousands of candidate pharmaceuticalcompounds must be evaluated. In addition, such a sensor would be usefulin monitoring bioprocesses and environmental pollution where theenhanced sensitivity of living cells to their environment can beexploited.

U.S. Ser. Nos. 09/033,462 and 09/260,963 describe arrays of cells thathave been confined to microcavities, such as wells on the ends of afiber optic bundle. These arrays may comprise either a single cell typeor mixtures of cell types. For the latter, since the cells are randomlyplaced in the microcavities, these applications provide for encoding ofcells through the use of membrane-binding fluorophores or the use ofcells that have been genetically altered to produce differentfluorophores, such as green fluorescent protein (GFP) variants.

However, a need exists for more robust and varied methods of encodingcell populations for the creation of cellular arrays.

SUMMARY OF THE INVENTION

Accordingly, the invention provides a cellular array for detecting theresponse of individual cells to at least one analyte of interest. Thearray includes a substrate comprising a plurality of discrete sites anda plurality of cells dispersed at said discrete sites, wherein each cellis encoded with at least one exogenous binding partner.

In addition the invention provides a method of making a cellular array.The method includes associating a population of cells with a populationof microspheres, such that each microsphere has at least one associatedcell. Preferably the population includes a first subpopulationcomprising a first cell type and at least a second subpopulationcomprising a second cell type, wherein the first subpopulation comprisesat least a first exogenous binding partner and the second subpopulationcomprises at least a second exogenous binding partner. The methodfurther includes distributing the population of microspheres ontodiscrete sites of a substrate, and identifying the location of at leastthe first and second exogenous binding partners.

In addition the invention provides a method of screening. The methodincludes contacting a candidate agent with a cellular array thatincludes a substrate comprising plurality of discrete sites and aplurality of cells dispersed at the discrete sites. Each cell is encodedwith at least one exogenous binding partner. The method further includesdetermining the effect of the candidate agent on the cells.

In addition the invention provides a method of screening that includescontacting cells with an array that includes a substrate comprising aplurality of discrete sites and a plurality of microspheres dispersed atthe sites, wherein each of the microspheres comprise a candidatebioactive agent.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In general, the invention provides for a biosensor, a biosensor array, abiosensor sensing system and sensing methods for the analysis ofchemical and biological materials. More particularly, the inventionprovides for biosensors and biosensor arrays, sensing apparatus andsensing methods which employ living cells and mixed populations ofliving cells for analysis of chemical and biological materials.

The biosensor array of the present invention comprises either amonoculture of living cells or randomly mixed populations of livingcells wherein each individual cell in the array is positioned on asubstrate at an optically-addressable, discrete site which preferablyaccommodate the size and shape of individual cells. In one embodiment,the discrete site comprises a microwell or microcavity which ispreformed to accommodate the size and shape of the individual cells. Insome embodiments, as is more fully outlined below, the cells may beassociated with microspheres that are loaded in the microwells. Thebiosensor array sensing method relies on the well known fact thatindividual cells, which are chemically or biologically stimulated by thepresence of a biological or chemical material in the cell environment,will respond by producing a change in the cell or cellular environmentwhich can be interrogated (generally optically) and detected within thecell itself or from an indicator compound, for example, a fluorophore,chromophore or dye, either attached to the cell, taken up in the cell,or added to the local cell environment. The biosensor of the presentinvention thus capitalizes on the ability of living cells to respond tobiologically significant compounds. Since the selectivity of livingcells for such compounds has considerable value and utility in candidateagent screening (e.g. drug screening) and analysis of complex biologicalfluids, the biosensor of the present invention offers distinctadvantages to high throughput screening of combinatorial libraries wherehundreds of thousands of candidate compounds must be evaluated.

By incorporating a biosensor into an optically interrogatable substrateor a fiber optic array, the innovation of the biosensor of the presentinvention is in providing for optical coupling of individual cellslocated at discrete substrate sites or microwells with discrete detectorelements, CCD cameras, or individual optical fibers in a fiber opticarray or bundle that are in optical communication with such devices. By“optical coupling”, “optical communication”, or “optical cooperation” orother grammatical equivalents herein is meant the capability of eitheroptically stimulating individual cells within the biosensor array withexcitation light or optically interrogating the optical response ofindividual cells within the array to analyses, by conveying light to andfrom individual cells located at discrete cites within the array usingeither conventional optical train elements or optical fibers. Sincetypical fiber optic arrays contain thousands of discrete individualfiber strands, the invention thus provides for the individual opticalcoupling and interrogation of thousands of cells within an array,thereby providing for a large number of independent cell responsemeasurements for each cell population within an array. Due to both thenumber of cell populations available and the correspondingly largenumber of individual cells within each cell population, a significantinnovation of the present invention is in providing for the summing andamplification of the characteristic optical response signatures ofmultiple independent measurements taken from cells within each cellpopulation, thereby improving the detection limit and sensitivity of thebiosensor.

An additional innovation of the present invention is that, by deployinga large number of cell populations within the array, and providing alarge number of individual cells in each population, the discriminatingcapabilities of the biosensor array toward biological or chemicalanalyses is significantly enhanced by providing for thousands of cellresponses from a large number of cell populations. This Feature directlymimics the actual behavior of the human olfactory system where thecombined signals from thousands of receptor cells, in each grouping ofnearly a thousand different receptor cell types found in the epitheliumlayer, none of which are particularly sensitive in themselves, lead to ahighly amplified sensory response to odors [see Kauer, et al, TrehdsNeurosci. 14: 79 (1991). One embodiment of the present invention thusmimics the evolutionary scent amplification process found in the humanolfactory system in order to significantly enhance biosensor arraysensitivity to analyses by summing the low-level responses of a largenumber of cells in the biosensor array. By summing the responses from anumber of cells at low analyte concentrations, a substantial improvementin signal-to-noise ratio can be achieved and a corresponding reductionin the detection limit of the biosensor array is obtained.

Thus, the present invention provides cellular arrays. By “array” hereinis meant a plurality of candidate agents in an array format; the size ofthe array will depend on the composition and end use of the array.Arrays containing from about 2 different bioactive agents (i.e.different beads) to many millions can be made, with very large fiberoptic arrays being possible. Generally, the array will comprise from twoto as many as a billion or more, depending on the size of the beads andthe substrate, as well as the end use of the array, thus very highdensity, high density, moderate density, low density and very lowdensity arrays may be made. Preferred ranges for very high densityarrays are from about 10,000,000 to about 2,000,000,000, with from about100,000,000 to about 1,000,000,000 being preferred (all numbers being insquare cm). High density arrays range about 100,000 to about 10,000,000,with from about 1,000,000 to about 5,000,000 being particularlypreferred. Moderate density arrays range from about 10,000 to about100,000 being particularly preferred, and from about 20,000 to about50,000 being especially preferred. Low density arrays are generally lessthan 10,000, with from about 1,000 to about 5,000 being preferred. Verylow density arrays are less than 1,000, with from about 10 to about 1000being preferred, and from about 100 to about 500 being particularlypreferred. In some embodiments, the compositions of the invention maynot be in array format; that is, for some embodiments, compositionscomprising a single bioactive agent may be made as well. In addition, insome arrays, multiple substrates may be used, either of different oridentical compositions. Thus for example, large arrays may comprise aplurality of smaller substrates.

In addition, one advantage of the present compositions is thatparticularly through the use of fiber optic technology, extremely highdensity arrays can be made. Thus for example, because beads of 200 μm orless (with beads of 200 nm possible) can be used, and very small fibersare known, it is possible to have as many as 250,000 or more (in someinstances, 1 million) different fibers and beads in a 1 mm² fiber opticbundle, with densities of greater than 25,000,000 individual beads andfibers (again, in some instances as many as 50-100 million) per 0.5 cm²obtainable.

By “substrate” or “solid support” or other grammatical equivalentsherein is meant any material that can be modified to contain discreteindividual sites appropriate for the attachment or association of beadsand is amenable to at least one detection method. As will be appreciatedby those in the art, the number of possible substrates is very large.Possible substrates include, but are not limited to, glass and modifiedor functionalized glass, plastics (including acrylics, polystyrene andcopolymers of styrene and other materials, polypropylene, polyethylene,polybutylene, polyurethanes, Teflon, etc.), polysaccharides, nylon ornitrocellulose, resins, silica or silica-based materials includingsilicon and modified silicon, carbon, metals, inorganic glasses,plastics, optical fiber bundles, and a variety of other polymers. Ingeneral, the substrates allow optical detection and do not themselvesappreciably fluoresces.

Generally the substrate is flat (planar), although as will beappreciated by those in the art, other configurations of substrates maybe used as well; for example, three dimensional configurations can beused, for example by embedding the cells in a porous block of materialsuch as plastic or agarose that allows solution access to the cells andusing a confocal microscope for detection. Similarly, the cells may beplaced on the inside surface of a tube, for flow-through sample analysisto minimize sample volume. Preferred substrates include optical fiberbundles as discussed below, and flat planar substrates such as glass,polystyrene and other plastics and acrylics.

In a preferred embodiment, the substrate is an optical fiber bundle orarray, as is generally described in U.S. Ser. Nos. 08/944,850 and08/519,062, PCT US98/05025, and PCT US98/09163, all of which areexpressly incorporated herein by reference. Preferred embodimentsutilize preformed unitary fiber optic arrays. By “preformed unitaryfiber optic array” herein is meant an array of discrete individual fiberoptic strands that are co-axially disposed and joined along theirlengths. The fiber strands are generally individually clad. However, onething that distinguished a preformed unitary array from other fiberoptic formats is that the fibers are not individually physicallymanipulatable; that is, one strand generally cannot be physicallyseparated at any point along its length from another fiber strand.

At least one surface of the substrate is modified to contain discrete,individual sites for later association of cells and/or microspheres.These sites may comprise physically altered sites, i.e. physicalconfigurations such as wells or small depressions in the substrate thatcan retain the cells, such that a cell can rest in the well, or the useof other forces (magnetic or compressive), or chemically altered oractive sites, such as chemically functionalized sites, electrostaticallyaltered sites, hydrophobically/hydrophilically functionalized sites,spots of adhesive, etc.

The sites may be a pattern, i.e. a regular design or configuration, orrandomly distributed. A preferred embodiment utilizes a regular patternof sites such that the sites may be addressed in the X-Y coordinateplane. “Pattern” in this sense includes a repeating unit cell,preferably one that allows a high density of cells on the substrate.However, it should be noted that these sites may not be discrete sites.That is, it is possible to use a uniform surface of adhesive or chemicalfunctionalities, for example, that allows the attachment of beads at anyposition. That is, the surface of the substrate is modified to allowattachment of the cells at individual sites, whether or not those sitesare contiguous or non-contiguous with other sites. Thus, the surface ofthe substrate may be modified such that discrete sites are formed thatcan only have a single associated cell, or alternatively, the surface ofthe substrate is modified and cells may go down anywhere, but they endup at discrete sites.

In a preferred embodiment, the surface of the substrate is modified tocontain wells, i.e. depressions in the surface of the substrate. Thismay be done as is generally known in the art using a variety oftechniques, including, but not limited to, photolithography, stampingtechniques, molding techniques and microetching techniques. As will beappreciated by those in the art, the technique used will depend on thecomposition and shape of the substrate.

In a preferred embodiment, physical alterations are made in a surface ofthe substrate to produce the sites. In a preferred embodiment, thesubstrate is a fiber optic bundle and the surface of the substrate is aterminal end of the fiber bundle, as is generally described in Ser. Nos.08/818,199 and 09/151,877, both of which are hereby expresslyincorporated by reference. In this embodiment, wells are made in aterminal or distal end of a fiber optic bundle comprising individualfibers. In this embodiment, the cores of the individual fibers areetched, with respect to the cladding, such that small wells ordepressions are formed at one end of the fibers. The required depth ofthe wells will depend on the size of the beads to be added to the wells.

In a preferred embodiment, an array of micrometer-sized wells is createdat the distal face of an optical imaging fiber by a selective etchingprocess which takes advantage of the difference in etch rates betweencore and cladding materials. This process has been previously disclosedby Pantano, et al., Chem. Mater. 8: 2832 (1996), and Walt, et al., inU.S. patent application Ser. No. 08/818,199. The etch reaction time andconditions are adjusted to achieve desired control over the resultantmicrowell size and volume. Microwells are thus sized to accommodate asingle cell of any desired cell type.

The sensor array design can accommodate a variety of cell sizes andconfigurations utilizing either commercially available optical fibersand fiber optic arrays or custom made fibers or fiber arrays. The majorrequirement in selecting candidate fibers or fiber optic arrays forfabricating sensors is that the individual fibers have etchable cores.

The method of fabricating microwells can be adapted to any fiber size soas to accommodate a wide range of cell sizes for incorporation intoappropriately sized microwells. For example, optical fibers having corediameters ranging from 1.6 to 100 um are commercially available fromeither Galileo ElectroOptics Corp. (Sturbridge, Mass.) or EdmundScientific (Barrington, N.J.). In addition, larger sizes are availableby custom order. Thus, appropriately sized fibers can be utilized tostudy such diverse cell sizes as E. coli, with a typical cell dimensionof 0.7 to 1.5 um, and mammalian neurons, with a cell dimension of up to150 um.

It is generally preferred that the cells are non-covalently associatedin the wells, although the wells may additionally be chemicallyfunctionalized as is generally described below, cross-linking agents maybe used, or a physical barrier may be used, i.e. a film or membrane overthe cells.

In a preferred embodiment, the surface of the substrate is modified tocontain chemically modified sites, that can be used to attach, eithercovalently or non-covalently, either cells or microspheres of theinvention to the discrete sites or locations on the substrate; however,in general, for covalent attachment, preferred embodiments utilize cellsassociated with microspheres that are chemically modified; that is,direct covalent attachment of cells to surfaces is generally notpreferred although it can be done. “Chemically modified sites” in thiscontext includes, but is not limited to, the addition of a pattern ofchemical functional groups including amino groups, carboxy groups, oxogroups and thiol groups, that can be used to for covalent attachment,which generally also contain corresponding reactive functional groups;the addition of a pattern of adhesive that can be used to bind themicrospheres (either by prior chemical functionalization for theaddition of the adhesive or direct addition of the adhesive); theaddition of a pattern of charged groups (similar to the chemicalfunctionalities) for the electrostatic attachment of the cells ormicrospheres, e.g. when the microspheres comprise charged groupsopposite to the sites; the addition of a pattern of chemical functionalgroups that renders the sites differentially hydrophobic or hydrophilic,such that the addition of similarly hydrophobic or hydrophilicmicrospheres under suitable experimental conditions will result inassociation of the microspheres to the sites on the basis ofhydroaffinity. For example, the use of hydrophobic sites withhydrophobic beads, in an aqueous system, drives the association of thebeads preferentially onto the sites. As outlined above, “pattern” inthis sense includes the use of a uniform treatment of the surface toallow attachment of the beads at discrete sites, as well as treatment ofthe surface resulting in discrete sites. As will be appreciated by thosein the art, this may be accomplished in a variety of ways.

In a preferred embodiment, biological modifications can be done to thesites to allow association of either cells or beads with cells. Theseinclude, but are not limited to, the use of binding ligands or bindingpartner pairs, including, but not limited to, antigen/antibody pairs,enzyme/substrate or inhibitor pairs, receptor-ligand pairs,carbohydrates and their binding partners (lectins, etc.).

In one embodiment, the interior surfaces of the microwells may be coatedwith a thin film or passivation layer of biologically compatiblematerial, similar to the biological modifications of the substrate asoutlined above. For example, materials known to support cell growth oradhesion may be used, including, but not limited to, fibronectin, anynumber of known polymers including collagen, polylysine and otherpolyamino acids, polyethylene glycol and polystyrene, growth factors,hormones, cytokines, etc. Preferred embodiments utilize cellularadhesion factors and proteins. Similarly, binding ligands as outlinedabove may be coated onto the surface of the wells. In addition, coatingsor films of metals such as a metal such as gold, platinum or palladiummay be employed. In an alternative embodiment, an indicator compound,for example, a fluorophore, a chromophore or dye, may be attached to themicrowell surface for detecting cell responses to chemical or biologicalstimulation.

The cellular array comprises individual sensor elements comprising cellsand/or beads. The arrays may comprise one type of cell or a plurality ofdifferent cell types. By “plurality” herein is meant at least two. Aswill be appreciated by those in the art, virtually any cell type andsize can be accommodated in fabricating the sensor of the presentinvention; when wells in the ends of fiber optic bundles are used, thecell size may be matched to the individual optical fiber optic corediameters. Virtually any naturally occurring or genetically engineered(i.e. containing exogenous nucleic acid) eukaryotic or procaryotic celltype may be used, with plants, invertebrates, bacteria and mammaliancells, including, but not limited to, primate, rodent and human cellsand cell lines being preferred, as well as mixtures of cell types.

In one embodiment, NIH 3T3 mouse fibroblast cells are employed. Thesecells are typically 15-20 um in size. Other cells types such as E. colibacteria, 1×3 um, staphylococcus bacteria, approximately 1 um, myoblastprecursors to skeletal muscle cells, 15-20 um, neutrophil white bloodcells, 10 um, lymphocyte white blood cells, 10 um, erythroblast redblood cells, 5 um, osteoblast bone cells, 15-20 um, chondrocytecartilage cells, 15-20 um, basophil white blood cells, 10 um, eosinophilwhite blood cells, 10 um, adipocyte fat cells, 20 um, invertebrateneurons (Helix aspera), 125 um, mammalian neurons, 4-140 um, oradrenomedullary cells, 13-16 um, melanocytes, 20 um, epithelial cells,20 um, or endothelial cells, 15-20 um, may be utilized as well.Additional other suitable cell types include, but are not limited to,tumor cells of all types (particularly melanoma, myeloid leukemia,carcinomas of the lung, breast, ovaries, colon, kidney, prostate,pancreas and testes), cardiomyocytes, endothelial cells, epithelialcells, lymphocytes (T-cell and B cell), mast cells, eosinophils,vascular intimal cells, hepatocytes, leukocytes including mononuclearleukocytes, stem cells such as haemopoetic, neural, skin, lung, kidney,liver and myocyte stem cells, osteoclasts, chondrocytes and otherconnective tissue cells, keratinocytes, melanocytes, liver cells, kidneycells, and adipocytes. Suitable cells also include known research cells,including, but not limited to, Jurkat T cells, NIH3T3 cells, CHO, COS,etc. A particularly useful source of cell lines may be found in ATCCCell Lines and Hybridomas (8^(th) ea., 1994), Bacteria andBacteriophages (19^(th) ed., 1996), Yeast (1995), Mycology and Botany(19^(th). ed, 1996), and Protists: Algae and Protozoa (18^(th) ed.,1993), available from American Type Culture Co. (Rockville, Md.), all ofwhich are herein incorporated by reference.

Cellular arrays comprising cells may be made by randomly dispersal ofthe cells into or onto the sites of the array. Cell populations areconventionally cultured with growth media which matches cell needs.Culture media is formulated according to either recipes provided by cellline providers, journal articles or reference texts. A particularlyuseful reference for media preparation is ATCC Quality Control/Methodsfor Cell Lines (2nd ed.), American Type Culture Co. (Rockville, Md.)which is herein incorporated by reference. After culturing, cells aretypically trypsinized using aseptic techniques to remove them from thecell culture dish and suspend them in growth media.

There are a variety of methods that can be employed to “load” thearrays. A preferred embodiment utilizes a cell suspension. In apreferred embodiment, prior to loading, the array surface may besonicated under vacuum in cell media for approximately 15 minutes toflush and fill the microwells with media. The cell suspension iscontacted with the substrate and allowed to interact for some period oftime to allow the suspended cells to settle into the wells and adhere tothe well bottom. The length of time required to fill the microwells isdependent only on the amount of time required for the cells to adhere tothe microwell bottom. Excess cells which were are accommodated by a wellcan be removed.

Once they are positioned within the microwells, cells will typicallyattach to microwell surfaces within 1-2 hours by protein contact. Cellculture media in the microwells may be periodically replenished byexposing the array to fresh media and allowing nutrients to diffuse intothe microwell cavities. Typically, cells will divide every twelve tofifteen hours. While the size of the microwells tends to confineindividual cells, the array will accommodate limited cell splitting overtime. Microwell volume will restrict cell splitting due to the well knowcell phenomenon of contact inhibition when cells are touching.

In a preferred embodiment, the cells are associated with microspheres.By “microspheres” or “beads” or “particles” or grammatical equivalentsherein is meant small discrete particles. The composition of the beadswill vary, depending on a variety of factors. Suitable bead compositionsinclude those used in peptide, nucleic acid and organic moietysynthesis, including, but not limited to, plastics, ceramics, glass,polystyrene, methylstyrene, acrylic polymers, paramagnetic materials,thoria sol, carbon graphite, titanium dioxide, agarose, latex orcross-linked dextrans such as Sepharose, cellulose, nylon, cross-linkedmicelles and Teflon may all be used. “Microsphere Detection Guide” fromBangs Laboratories, Fishers, Ind. is a helpful guide.

The beads need not be spherical; irregular particles may be used. Inaddition, the beads may be porous, thus increasing the surface area ofthe bead available for either binding partner attachment or cellassociation. The bead sizes range from nanometers, i.e. 100 nm, tomillimeters, i.e. 1 mm, with beads of at least m150 micron beingpreferred, and from about 0.2 micron to about 200 microns being morepreferred, and from about 0.5 to about 5 micron being particularlypreferred, although in some embodiments smaller beads may be used.

It should be noted that a key component of the invention is the use of asubstrate/bead pairing that allows the association or attachment of thebeads at discrete sites on the surface of the substrate, such that thebeads do not move during the course of the assay.

In a preferred embodiment, porous beads are used, such that the cellsmay infuse or be trapped within the beads, although care should be takento allow media access to the cells. Alternatively, cells may be grown onthe exposed surfaces of beads, including porous beads, using techniquesknown in the art for planar substrates.

Similarly, cells may be embedded in a bead matrix such as agarose, andmicroparticles generated with the resulting material.

In one embodiment, the cells are associated with the beads as outlinedabove for chemical or biological functionalization.

When cells are associated with microspheres, the cellular arrays can befabricated as previously described for microsphere arrays; see PCTUS98/05025 for example, hereby expressly incorporated by reference. Ingeneral, the arrays are made by adding a solution or slurry comprisingthe cell-bead compositions (sometimes referred to herein as “particles”)to a surface containing the sites for association of the particles. Thismay be done in a variety of buffers, including aqueous and organicsolvents, and mixtures. The solvent can evaporate, and excess particlesremoved.

In a preferred embodiment, when non-covalent methods are used toassociate the particles on the array, a novel method of loading theparticles onto the array is used. This method comprises exposing thearray to a solution of particles and then applying energy, e.g.agitating or vibrating the mixture. This results in an array comprisingmore tightly associated particles, as the agitation is done withsufficient energy to cause weakly-associated particles to fall off (orout, in the case of wells). These sites are then available to bind adifferent particle. In this way, particles that exhibit a high affinityfor the sites are selected. Arrays made in this way have two mainadvantages as compared to a more static loading: first of all, a higherpercentage of the sites can be filled easily, and secondly, the arraysthus loaded show a substantial decrease in particle loss during assays.Thus, in a preferred embodiment, these methods are used to generatearrays that have at least about 50% of the sites filled, with at leastabout 75% being preferred, and at least about 90% being particularlypreferred. Similarly, arrays generated in this manner preferably loseless than about 20% of the particles during an assay, with less thanabout 10% being preferred and less than about 5% being particularlypreferred.

In this embodiment, the substrate comprising the surface with thediscrete sites is immersed into a solution comprising the particles(beads, cells, etc.). The surface may comprise wells, as is describedherein, or other types of sites on a patterned surface such that thereis a differential affinity for the sites. This differential affinityresults in a competitive process, such that particles that willassociate more tightly are selected. Preferably, the entire surface tobe “loaded” with particles is in fluid contact with the solution. Thissolution is generally a slurry ranging from about 10,000:1beads:solution (vol:vol) to 1:1. Generally, the solution can compriseany number of reagents, including aqueous buffers, organic solvents,salts, other reagent components, etc. In addition, the solutionpreferably comprises an excess of particles; that is, there are moreparticles than sites on the array. Preferred embodiments utilizetwo-fold to billion-fold excess of particles.

The immersion can mimic the assay conditions; for example, if the arrayis to be “dipped” from above into a microtiter plate comprising samples,this configuration can be repeated for the loading, thus minimizing theparticles that are likely to fall out due to gravity.

Once the surface has been immersed, the substrate, the solution, or bothare subjected to a competitive process, whereby the particles with loweraffinity can be disassociated from the substrate and replaced byparticles exhibiting a higher affinity to the site. This competitiveprocess is done by the introduction of energy, in the form of heat,sonication, stirring or mixing, vibrating or agitating the solution orsubstrate, or both. Care should be taken to avoid damaging the cells.

A preferred embodiment utilizes agitation or vibration. In general, theamount of manipulation of the substrate is minimized to prevent damageto the array; thus, preferred embodiments utilize the agitation of thesolution rather than the array, although either will work. As will beappreciated by those in the art, this agitation can take on any numberof forms, with a preferred embodiment utilizing microtiter platescomprising bead solutions being agitated using microtiter plate shakers.

The agitation proceeds for a period of time sufficient to load the arrayto a desired fill. Depending on the size and concentration of theparticles and the size of the array, this time may range from about 1second to days, with from about 1 minute to about 24 hours beingpreferred.

It should be noted that not all sites of an array may comprise a cell;that is, there may be some sites on the substrate surface which areempty. In addition, there may be some sites that contain more than onecell. This is acceptable if for example beads are used and only onebead, that may comprise a plurality of cells of a single cell type, isassociated with the site.

In addition, cell viability tests such as are known in the art may bedone after loading of the arrays. These include, but are not limited to,the use of pH indicators and well known dyes for cell viability; see theMolecular Probes Handbook, supra.

In contrast to the systems described in U.S. Ser. Nos. 09/033,462 and09/260,963, the present invention provides for novel and robust methodsof encoding the individual cells and cell populations in the array formaintaining cell type identity and location where randomly mixedpopulations of cells are employed. Cells may be encoded prior todisposing them in the microwells (or at the sites) or, alternatively,following placement in the microwells. Although cell populations may berandomly mixed together, this innovative feature provides for theidentity and location of each cell type to be determined via acharacteristic optical response signature when the cell array is eitherilluminated by excitation light energy or, alternatively, subjected tobiological stimuli.

The invention provides for novel encoding methods, including, but notlimited to, the use of identifier binding ligands (IBLs), preferablythat bind to antibodies or fluorophores. That is, by using uniqueligands that will bind a unique partner, the identity of the cell typemay be elucidated. For example, cell surface receptors specific toimmunological cells can be used with uniquely labeled ligands todistinguish from cells not carrying the receptor; upon binding of theligand to the receptor, the label is now present at the array site andcan be detected; antibodies to cell surface receptors will also workwell. The IBLs may be either endogenous (native to the cell) orexogenous (not native to the cell, e.g. recombinantly introduced).

By “identifier binding ligands” or “IBLs” herein is meant a compoundthat will specifically bind a corresponding decoder binding ligand (DBL)to facilitate the elucidation of the identity of the cell. That is, theIBL and the corresponding DBL form a binding partner pair. By“specifically bind” herein is meant that the IBL binds its DBL withspecificity sufficient to differentiate between the corresponding DBLand other DBLs (that is, DBLs for other IBLs), or other components orcontaminants of the system. The binding should be sufficient to remainbound under the conditions of the decoding step, including wash steps toremove non-specific binding. In some embodiments, for example when theIBLs and corresponding DBLs are proteins or nucleic acids, thedissociation constants of the IBL to its DBL will be less than about10⁻⁴-10⁻⁶ M⁻¹, with less than about 10⁻⁵ to 10⁻⁹ M⁻¹ being preferred andless than about 10⁻⁷-10⁻⁹ M⁻¹ being particularly preferred.

IBL-DBL binding pairs are known or can be readily found using knowntechniques. For example, when the IBL is a protein, the DBLs includeproteins (particularly including antibodies or fragments thereof (FAbs,etc.)) or small molecules, or vice versa (the IBL is an antibody and theDBL is a protein). Metal ion-metal ion ligands or chelators pairs arealso useful. Antigen-antibody pairs, enzymes and substrates orinhibitors, other protein-protein interacting pairs, receptor-ligands,complementary nucleic acids (including nucleic acid molecules that formtriple helices), and carbohydrates and their binding partners are alsosuitable binding pairs. Nucleic acid—nucleic acid binding proteins pairsare also useful, including single-stranded or double-stranded nucleicacid binding proteins, and small molecule nucleic acid binding agents.Similarly, as is generally described in U.S. Pat. Nos. 5,270,163,5,475,096, 5,567,588, 5,595,877, 5,637,459, 5,683,867,5,705,337, andrelated patents, hereby incorporated by reference, nucleic acid“aptamers” can be developed for binding to virtually any target; such anaptamer-target pair can be used as the IBL-DBL pair. Similarly, there isa wide body of literature relating to the development of binding pairsbased on combinatorial chemistry methods.

In a preferred embodiment, each subpopulation of cells (e.g. each celltype) comprises a plurality of different IBLs. By using a plurality ofdifferent IBLs to encode each cell, the number of possible unique codesis substantially increased. That is, by using one unique IBL per cell,the size of the array will be the number of unique IBLs (assuming no“reuse” occurs, as outlined below). That is, 20 different cell types canbe distinguished (assuming sufficient specificity and thus a lack ofcross-reactivity) using 20 different IBLs. However, by using a pluralityof different IBLs per cell, n, the size of the array can be increased to2^(n), when the presence or absence of each IBL is used as theindicator. For example, the assignment of 10 IBLs per bead generates a10 bit binary code, where each bit can be designated as “1” (IBL ispresent) or “0” (IBL is absent). A 10 bit binary code has 2¹⁰ possiblevariants. However, as is more fully discussed below, the size of thearray may be further increased if another parameter is included such asconcentration or intensity; thus for example, if two differentconcentrations of the IBL are used (e.g. two different expressionlevels), then the array size increases as 3^(n). Thus, in thisembodiment, each individual cell in the array is assigned a combinationof IBLs.

In addition, the use of different concentrations or densities of IBLsallows a “reuse” of sorts. If, for example, a first cell type comprisesan expression vector that results in a 1× expression level of IBL, and asecond cell type comprises a different construct that gives a 10×expression level of IBL, using saturating concentrations of thecorresponding labeled DBL allows the user to distinguish between the twocell types.

As outlined herein, the IBL-DBL pair may be selected from any number ofbinding partner pairs, with antigen-antibody pairs, other cell surfacereceptor-ligand pairs (including for example, insulin receptor,insulin-like growth factor receptor, growth hormone receptor, glucosetransporters (particularly GLUT 4 receptor), transferrin receptor,epidermal growth factor receptor, low density lipoprotein receptor, highdensity lipoprotein receptor, epidermal growth factor receptor, leptinreceptor, interleukin receptors including IL-1, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-15, and IL-17 receptors,human growth hormone receptor, VEGF receptor, PDGF receptor, EPOreceptor, TPO receptor, ciliary neurotrophic factor receptor, prolactinreceptor, and T-cell receptors; all of which have associated ligands),and fluorophore binding peptides as is known in the art; see RozinovChem. Biol. 5: 713 (1998) and Whitney et al., Nature Biotech. 16: 1329(1998), both of which are expressly incorporated by reference.

In a preferred embodiment, the IBL is expressed on the surface of thecell, to allow access of binding partners such as antibodies orfluorophores. This is done as is generally known in the art, usingspecific signal sequences or membrane anchoring sequences, including butnot limited to, Particularly preferred membrane-anchoring sequencesinclude, but are not limited to, those derived from CD8, ICAM-2, IL-8R,CD4 and LFA-1; the GPI anchor, which results in a covalent bond betweenthe molecule and the lipid bilayer via a glycosyl-phosphatidylinositolbond; myristylation sequences, which serve as membrane anchoringsequences since the myristylation of c-src recruits it to the plasmamembrane; and palmitoylation sequences.

However, in some embodiments the IBLs are located within the cell, andthe DBLs are introduced into the cell as well.

In general, cells are transformed using techniques well known in theart. Preferred embodiments utilize a variety of vectors and constructswith phage vectors being particularly preferred, including, but notlimited to, retroviruses, adenoviruses, FIV, lentiviruses, etc.

When IBLs (e.g. codes) are introduced to cells by way of recombinanttechniques, preferred embodiments utilize the delivery of additionalnucleic acids, which can serve either as candidate agents, as outlinedherein, or for more specific purposes. For example, the additionalnucleic acid could be a new gene added to a cell, or an antisense gene.Linking new nucleic acid with a “code” allows the tracking of changeswithin the cell; this tracking can create a functional assay fordetermining the effectors of a gene (including non-coding regions) whenintroduced to a cell.

In a preferred embodiment, when cells are first associated with beads toform “particles” as outlined herein, the IBLs may be found on the beads.That is, subpopulations of cell types are associated with beads thatcomprise IBLs that can be used to locate these beads.

In a preferred embodiment, the location of the different cell types onthe array is determined using decoder binding ligands (DBLs). Asoutlined above, DBLs are binding ligands that will bind to identifierbinding ligands.

In a preferred embodiment, the DBLs are either directly or indirectlylabeled. By “labeled” herein is meant that a compound has at least oneelement, isotope or chemical compound attached to enable the detectionof the compound. In general, labels fall into three classes: a) isotopiclabels, which may be radioactive or heavy isotopes; b) magnetic,electrical, thermal; and c) colored or luminescent dyes; although labelsinclude enzymes and particles such as magnetic particles as well.Preferred labels include luminescent labels. In a preferred embodiment,the DBL is directly labeled, that is, the DBL comprises a label. In analternate embodiment, the DBL is indirectly labeled; that is, a labelingbinding ligand (LBL) that will bind to the DBL is used. In thisembodiment, the labeling binding ligand-DBL pair can be as describedabove for IBL-DBL pairs. Suitable labels include, but are not limitedto, fluorescent lanthanide complexes, including those of Europium andTerbium, fluorescein, rhodamine, tetramethylrhodamine, eosin,erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green,stilbene, Lucifer Yellow, Cascade Blue, Texas Red, FITC, PE, cy3, cy5and others described in the 6th Edition of the Molecular Probes Handbookby Richard P. Haugland, hereby expressly incorporated by reference.

Accordingly, the identification of the location of the individual cells(or subpopulations of cells) is done using one or more decoding stepscomprising a binding between the labeled DBL and the IBL. Afterdecoding, the DBLs can be removed and the array can be used; however, insome circumstances, the removal of the DBL is not required (although itmay be desirable in some circumstances). In addition, as outlinedherein, decoding may be done either before the array is used in anassay, during the assay, or after the assay.

In one embodiment, a single decoding step is done. In this embodiment,each DBL is labeled with a unique label, such that the number of uniquelabels is equal to or greater than the number of different cell types(although in some cases, “reuse” of the unique labels can be done, asdescribed herein). For each IBL, a DBL is made that will specificallybind to it and contains a unique label, for example one or morefluorochromes. Thus, the identity of each DBL, both its composition andits label, is known. Then, by adding the DBLs to the array containingthe cells under conditions which allow the formation of bindingcomplexes between the DBLs and the IBLs, the location of each DBL can beelucidated. This allows the identification of the location of eachcells; the random array has been decoded. The DBLs can then be removed,if necessary, and the target sample applied.

In a preferred embodiment, the number of unique labels is less than thenumber of unique cell types, and thus a sequential series of decodingsteps are used, as is outlined in U.S. Ser. Nos. 09/189,543 and09/344,526, hereby incorporated by reference.

In one embodiment, the DBLs are labeled in situ; that is, they need notbe labeled prior to the decoding reaction.

In a preferred embodiment, the DBLs may be reused by having somesubpopulations of cells comprise optical signatures as is generallydescribed in U.S. Ser. Nos. 09/033,462 and 09/260,963. In a preferredembodiment, the optical signature is generally a mixture of reporterdyes, preferably flourescent. By varying both the composition of themixture (i.e. the ratio of one dye to another) and the concentration ofthe dye (leading to differences in signal intensity), matrices of uniqueoptical signatures may be generated.

Once made and decoded as required, the cellular arrays of the presentinvention find use in a number of applications. The biosensor, biosensorarray, sensing apparatus and sensing method of the present invention canbe applied to a large variety of conventional assays for screening anddetection purposes. The biosensor may be configured for virtually anyassay and offers a distinct advantage for high throughput screeningwhere a plurality of encoded cell populations, which have utility inparticular assays or are genetically engineered cell to provide uniqueresponses to analytes, may be employed in a single sensor array forconducting a large number of assays simultaneously on a small sample.The biosensor array thus provides both for tremendous efficiencies inscreening large combinatorial libraries and allows conduction of a largenumber of assays on extremely small sample volumes, such as biologicallyimportant molecules synthesized on micron sized beads. The biosensor ofthe present invention can be applied to virtually any analytemeasurements where there is a detectable cell response to the analytedue to biological stimulation.

The biosensor array and method of the present invention utilizes theunique ability of living cell populations to respond to biologicallysignificant compounds in a characteristic and detectable manner. Sincethe selectivity of living cells for such compounds has considerablevalue and utility in drug screening and analysis of complex biologicalfluids, a biosensor which makes use of the unique characteristics ofliving cell populations offers distinct advantages in high throughputscreening of combinatorial libraries where hundreds of thousands ofcandidate pharmaceutical compounds must be evaluated. In addition, sucha biosensor and sensing method can be utilized for either off-linemonitoring of bioprocesses or in situ monitoring of environmentalpollutants where the enhanced sensitivity of living cells to their localenvironment can be exploited.

Thus, the present invention provides methods for detecting the responsesof individual cells to analyses of interest. By “analyte of interest” or“target analyte” or “candidate bioactive agent” or “candidate drug” orgrammatical equivalents herein is meant any molecule, e.g., protein,oligopeptide, small organic molecule, polysaccharide, polynucleotide,etc., to be tested for the ability to directly or indirectly altering acellular phenotype, including its optical properties. Generally aplurality of assay mixtures are run in parallel with different agentconcentrations to obtain a differential response to the variousconcentrations. Typically, one of these concentrations serves as anegative control, i.e., at zero concentration or below the level ofdetection.

Analytes encompass numerous chemical classes, though typically they areorganic molecules, preferably small organic compounds having a molecularweight of more than 100 and less than about 2,500 daltons. Analytescomprise functional groups necessary for structural interaction withproteins, particularly hydrogen bonding, and typically include at leastan amine, carbonyl, hydroxyl or carboxyl group, preferably at least twoof the functional chemical groups. The candidate agents often comprisecyclical carbon or heterocyclic structures and/or aromatic orpolyaromatic structures substituted with one or more of the abovefunctional groups. Candidate agents are also found among biomoleculesincluding peptides, saccharides, fatty acids, steroids, purines,pyrimidines, derivatives, structural analogs or combinations thereof.Particularly preferred are peptides.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides. Alternatively, libraries of natural compounds in theform of bacterial, fungal, plant and animal extracts are available orreadily produced. Additionally, natural or synthetically producedlibraries and compounds are readily modified through conventionalchemical, physical and biochemical means. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification to producestructural analogs.

In a preferred embodiment, the candidate bioactive agents are naturallyoccurring proteins or fragments of naturally occurring proteins. Thus,for example, cellular extracts containing proteins, or random ordirected digests of proteinaceous cellular extracts, may be used. Inthis way libraries of procaryotic and eucaryotic proteins may be madefor screening in the methods of the invention. Particularly preferred inthis embodiment are libraries of bacterial, fungal, viral, and mammalianproteins, with the latter being preferred, and human proteins beingespecially preferred.

The peptides may be digests of naturally occurring proteins as isoutlined above, random peptides, or “biased” random peptides. By“randomized” or grammatical equivalents herein is meant that eachnucleic acid and peptide consists of essentially random nucleotides andamino acids, respectively. Since generally these random peptides (ornucleic acids, discussed below) are chemically synthesized, they mayincorporate any nucleotide or amino acid at any position. The syntheticprocess can be designed to generate randomized proteins or nucleicacids, to allow the formation of all or most of the possiblecombinations over the length of the sequence, thus forming a library ofrandomized candidate bioactive proteinaceous agents.

In one embodiment, the library is fully randomized, with no sequencepreferences or constants at any position. In a preferred embodiment, thelibrary is biased. That is, some positions within the sequence areeither held constant, or are selected from a limited number ofpossibilities. For example, in a preferred embodiment, the nucleotidesor amino acid residues are randomized within a defined class, forexample, of hydrophobic amino acids, hydrophilic residues, stericallybiased (either small or large) residues, towards the creation of nucleicacid binding domains, the creation of cysteines, for cross-linking,prolines for SH-3 domains, serines, threonines, tyrosines or histidinesfor phosphorylation sites, etc., or to purines, etc.

As described above generally for proteins, nucleic acid candidatebioactive agents may be naturally occurring nucleic acids, randomnucleic acids, or “biased” random nucleic acids. For example, digests ofprocaryotic or eucaryotic genomes may be used as is outlined above forproteins. By “nucleic acid” or “oligonucleotide” or grammaticalequivalents herein means at least two nucleotides covalently linkedtogether. A nucleic acid of the present invention will generally containphosphodiester bonds, although in some cases, as outlined below, nucleicacid analogs are included that may have alternate backbones, comprising,for example, phosphoramide (Beaucage et al., Tetrahedron 49(10): 1925(1993) and references therein; Letsinger, J. Org. Chem. 35: 3800 (1970);Sprinzl et al., Eur. J. Biochem. 81: 579 (1977); Letsinger et al., Nucl.Acids Res. 14: 3487 (1986); Sawai et al, Chem. Lett. 805 (1984),Letsinger et al., J. Am. Chem. Soc. 110: 4470 (1988); and Pauwels etal., Chemica Scripta 26: 141 91986)), phosphorothioate (Mag et al.,Nucleic Acids Res. 19: 1437 (1991); and U.S. Pat. No. 5,644,048),phosphorodithioate (Briu et al., J. Am. Chem. Soc. 111: 2321 (1989),O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides andAnalogues: A Practical Approach, Oxford University Press), and peptidenucleic acid backbones and linkages (see Egholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed. Engl. 31: 1008 (1992);Nielsen, Nature, 365: 566 (1993); Carlsson et al., Nature 380: 207(1996), all of which are incorporated by reference). Other analognucleic acids include those with positive backbones (Denpcy et al.,Proc. Natl. Acad. Sci. USA 92: 6097 (1995); non-ionic backbones (U.S.Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863;Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30: 423 (1991);Letsinger et al., J. Am. Chem. Soc. 110: 4470 (1988); Letsinger et al.,Nucleoside & Nucleotide 13: 1597 (1994); Chapters 2 and 3, ASC SymposiumSeries 580, “Carbohydrate Modifications in Antisense Research”, Ed. Y.S. Sanghui and P. Dan Cook; Mesmaeker et al., Bioorganic & MedicinalChem. Lett. 4: 395 (1994); Jeffs et al., J. Biomolecular NMR 34: 17(1994); Tetrahedron Lett. 37: 743 (1996)) and non-ribose backbones,including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, andChapters 6 and 7, ASC Symposium Series 580, “Carbohydrate Modificationsin Antisense Research”, Ed. Y. S. Sanghui and P. Dan Cook. Nucleic acidscontaining one or more carbocyclic sugars are also included within thedefinition of nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995)pp 169-176). Several nucleic acid analogs are described in Rawls, C & ENews Jun. 2, 1997 page 35. All of these references are hereby expresslyincorporated by reference. These modifications of the ribose-phosphatebackbone may be done to facilitate the addition of labels, or toincrease the stability and half-life of such molecules in physiologicalenvironments.

As will be appreciated by those in the art, all of these nucleic acidanalogs may find use in the present invention. In addition, mixtures ofnaturally occurring nucleic acids and analogs can be made;alternatively, mixtures of different nucleic acid analogs, and mixturesof naturally occurring nucleic acids and analogs may be made.

The nucleic acids may be single stranded or double stranded, asspecified, or contain portions of both double stranded or singlestranded sequence. The nucleic acid may be DNA, both genomic and cDNA,RNA or a hybrid, where the nucleic acid contains any combination ofdeoxyribo- and ribo-nucleotides, and any combination of bases, includinguracil, adenine, thymine, cytosine, guanine, inosine, xathaninehypoxathanine, isocytosine, isoguanine, etc. As used herein, the term“nucleoside” includes nucleotides as well as nucleoside and nucleotideanalogs, and modified nucleosides such as amino modified nucleosides. Inaddition, “nucleoside” includes non-naturally occurring analogstructures. Thus for example the individual units of a peptide nucleicacid, each containing a base, are referred to herein as a nucleoside.

In a preferred embodiment, the candidate bioactive agents are organicchemical moieties, a wide variety of which are available in theliterature.

While the examples below provide a variety of specific assays which maybe useful in configuring and employing the biosensor array and method ofthe present invention, they are not intended to limit either the scopeof applications envisioned or the broad range of sensing methods whichcan be employed with a plurality of cell populations with the biosensorof the present invention.

In one embodiment, the biosensor array can be employed for remotelymonitoring redox states of individual cells or cell populations inbioprocesses. For example, NADH dependent fluorescence can be measuredin bacteria, fungi, plant or animal cells. NAD(P)/NAD(P)H can bemeasured to monitor changes from aerobic to anaerobic metabolism infermentation processes using the method disclosed by Luong, et al., inPractical Fluorescence, G. Guilbault ed., Marcel Dekker (New York,1990).

Alternatively, the biosensor array may be employed for in situmonitoring of cellular processes in response to environmentalcontaminants by incorporating the method disclosed by Hughes, et al.,Analytica Chimica Acta 307: 393 (1995) to provide for distinguishablecell population responses within an array. In this method, micron-sizedspheres, impregnated with a fluorophore and modified on the surface witha fluorogenic enzyme probe, are ingested by cells and enzymatic activityoccurs at the sphere surface, producing a detectable fluorescent signal.

In yet another embodiment, the biosensor array can be employed withgenetically engineered bioluminescent bacteria for in situ monitoringand optical sensing of metallic compounds. For example, cell populationresponses to antimonite and arsenite may be utilized by incorporatingthe method disclosed in Ramanathan, et al., Anal Chem. 69: 3380 (1997)into cell populations within the biosensor array. With this method, cellplasmid regulates the expression of bacterial luciferase depending onthe metal concentration.

In another embodiment, the cell populations within the biosensor arraycan be encoded with ATP dependent luminescent proteins, for examplefirefly luciferase, which are injected into rat hepatocytes forpathological studies according to the method disclosed by Koop, et al.,Biochem. J. 295: 165 (1993). These cells exhibit a decrease incytoplasmic ATP when exposed to pathological insults and changes influorescence directly relate to the extent of metabolic poisoning in thecell.

In one embodiment, the cell populations within the biosensor array canbe encoded with green fluorescent protein [see T. Gura, Science 276:1989 (1997); Niswender, et al., J. Microscopy 180(2): 109 (1995);Cubitt, et al., TIBS 20: 448 (1995); Miyawaki, et al., Nature 388: 882(1997)]. Several genetically-engineered mutants of GFP are availablewhich have distinguishable fluorescence emission wavelengths. Theseproteins have additional utility as fluorescing indicators of geneexpression and Ca⁺ levels within cells.

In an additional embodiment, the biosensor array can be used inmeasurements of cell proliferation by in situ monitoring of calciumlevels and calcium oscillations in single cells using fluorescentmarkers, such as aequorin or fura-2, according to the method disclosedby Cobbold, et al., Cell Biology 1: 311 (1990).

As will be appreciated by those in the art, the assays of the inventionmay be run in a wide variety of ways and for a wide variety of purposes.For example, the cells may be used as a detection system for aparticular analyte; the cells undergo a characteristic opticallydetectable change in the presence of a particular analyte.Alternatively, the cells may be used to screen drug candidate librariesfor the ability to alter a cellular phenotype that is opticallydetectable. For example, the expression of a therapeutically relevantcell surface receptor may be increased such that the receptor can nowbind a fluorescent ligand; similarly a therapeutically relevant enzymemay now be activated such that a fluorescent reaction product isgenerated. Alternatively, the candidate agent(s) may be introduced, andthen a secondary marker is added to detect changes in cellular states;for example, apoptosis can be detected using fluorescent annexin, etc.See the Molecular Probes Handbook, supra. In this way any modulation,including both increases and decreases, may be monitored. Similarly, theuse of reporter genes such as green fluorescent proteins and derivativesthereof facilitates high throughput screening for relevant analyteinteractions, through the use of inducible promoters, for example.

Generally, in a preferred embodiment, a candidate bioactive agent isadded to the cells prior to analysis, and the cells allowed to incubatefor some period of time. By “administration” or “contacting” herein ismeant that the candidate agent is added to the cells in such a manner asto allow the agent to act upon the cell, whether by uptake andintracellular action, or by action at the cell surface. In someembodiments, nucleic acid encoding a proteinaceous candidate agent (i.e.a peptide) may be put into a viral construct such as a retroviralconstruct and added to the cell, such that expression of the peptideagent is accomplished; see PCT US97/01019, hereby expressly incorporatedby reference.

Once the candidate agent has been administered to the cells, the cellscan be washed if desired and are allowed to incubate under preferablyphysiological conditions for some period of time.

The reactions outlined herein may be accomplished in a variety of ways,as will be appreciated by those in the art. Components of the reactionmay be added simultaneously, or sequentially, in any order. In addition,the reaction may include a variety of other reagents may be included inthe assays. These include reagents like salts, buffers, neutralproteins, e.g. albumin, detergents, etc which may be used to facilitateoptimal detection, and/or reduce non-specific or backgroundinteractions. Also reagents that otherwise improve the efficiency of theassay, such as protease inhibitors, nuclease inhibitors, antimicrobialagents, etc., may be used.

In general, at least one component of the assay is labeled. By “labeled”herein is meant that the compound is either directly or indirectlylabeled with a label which provides a detectable signal, e.g.radioisotope, fluorescers, enzyme, antibodies, particles such asmagnetic particles, chemiluminescers, or specific binding molecules,etc. Specific binding molecules include pairs, such as biotin andstreptavidin, digoxin and antidigoxin etc. For the specific bindingmembers, the complementary member would normally be labeled with amolecule which provides for detection, in accordance with knownprocedures, as outlined above. The label can directly or indirectlyprovide a detectable signal.

Once the assay is run, the data is analyzed to determine theexperimental outcome, i.e. either the presence or absence of a targetanalyte, the effect of a candidate agent on a cellular phenotype, etc.This is generally done using a computer.

In this way, bioactive agents are identified. Compounds withpharmacological activity are able to alter a cellular phenotype. Thecompounds having the desired pharmacological activity may beadministered in a physiologically acceptable carrier to a host, aspreviously described, he agents may be administered in a variety ofways, orally, parenterally e.g., subcutaneously, intraperitoneally,intravascularly, etc. Depending upon the manner of introduction, thecompounds may be formulated in a variety of ways. The concentration oftherapeutically active compound in the formulation may vary from about0.1-100 wt. %.

The pharmaceutical compositions can be prepared in various forms, suchas granules, tablets, pills, suppositories, capsules, suspensions,salves, lotions and the like. Pharmaceutical grade organic or inorganiccarriers and/or diluents suitable for oral and topical use can be usedto make up compositions containing the therapeutically-active compounds.Diluents known to the art include aqueous media, vegetable and animaloils and fats. Stabilizing agents, wetting and emulsifying agents, saltsfor varying the osmotic pressure or buffers for securing an adequate pHvalue, and skin penetration enhancers can be used as auxiliary agents.

Accordingly, individual cells or arrays of cells and cell populationsmay be optically interrogated and cell responses to analyses may bemeasured by conventional optical methods and instrumentation that areknown to those skilled in the art. Cells may be optically interrogatedwith any suitable excitation light energy source, such as arc lamps,lasers, or light emitting diodes, that are capable of producing light atan appropriate wavelength for exciting dye indicators that may beemployed for encoding cell populations or for responding to analyses ofinterest. The optical responses of individual cells or cell populationsmay be monitored and measured with any suitable optical detection means,including, but not limited to film or conventional optical detectors,such as photoresistors, photomultiplier tubes, photodiodes, or chargecoupled device (CCD) cameras. In a preferred embodiment, CCD cameras areemployed to capture fluorescent images of the biosensor array fordetecting responses of each cell and various cell subpopulations toanalyses. Where external optical stimulation of cells is required toelicit an optical response, conventional light sources such as arclamps, photodiodes, or lasers may be employed for excitation lightenergy. Cell responses may be monitored by conventional detectors suchas photomultiplier tubes, photodiodes, photoresistors or charge coupleddevice (CCD) cameras. Conventional optical train components, such aslenses, filters, beam splitters, dichroics, prisms and mirrors may beemployed to convey light to an from such light sources and detectorseither to discrete substrate sites or through optical fiber strands tomicrowells that contain individual cells. The principal requirement forany particular optical apparatus configuration that is employed inoptical measurements is that the combination of optical componentsprovide for optically coupling the cells in the array to detectors andlight sources. While a particular apparatus configuration that wasemployed in experimental optical measurements is described below, otherconfigurations may also be employed which are functionally equivalentand appropriate suited for a particular measurement requirement. In thisembodiment, both individual cell responses and a captured image of thearray response may be employed for detecting analytes.

In summary, the biosensor array and sensing method of the presentinvention offers many distinct advantages in overcoming the limitationsof prior art devices. The sensor arrays are easily fabricated fromcommercially available optical imaging fibers to yield a cost effective,high density, precisely formed, biosensor array without requiring anysophisticated machining or forming process. Since optical fibers andfiber optic arrays are available in a wide variety of fiber corediameters, most cell types and sizes may be accommodated in by thedevice and method of the present invention. In addition, cells can bereadily dispersed into the microwell array in random fashion with noneed for physical indexing or scanning to locate individual cells orcell populations due to the innovative cell encoding technique. Sensingmethods and sensing systems which employ the biosensor and sensor arrayof the present invention avoids many of the limitations in manipulatingcells encountered with prior art devices. Once cells are placed withinthe microwells of the array, conventional imaging systems and methodswhich employ an imaging camera and conventional optics, can monitor theresponse of thousands of cells simultaneously, eliminating requirementsfor mechanical scanning mechanisms. Analysis of measurement data isfurther facilitated by implementing commercially available imagingsoftware to process images of the biosensor array using patternrecognition techniques combined with neural network and otherstatistical methods.

The biosensor array and sensing method of the present invention may beemployed for a number of useful analytical applications where individualcells, which are chemically or biologically stimulated by the presenceof a biological or chemical material in the local cell environment, willrespond to their environment by producing an optically detectableresponse either due to the presence of an appropriate indicator or dueto the characteristic optical response of particular cell types whichexhibit either natural or genetically-engineered chemiluminescence orbioluminescence. The biosensor array and method of the present inventionthus capitalizes on the ability of living cells to respond tobiologically significant compounds. Since the selectivity of livingcells for such compounds has considerable value and utility in drugscreening and analysis of complex biological fluids, the biosensor ofthe present invention offers distinct advantages to high throughputscreening of combinatorial libraries where hundreds of thousands ofcandidate compounds must be evaluated.

The present invention also provides methods of using microsphere arraysfor screening candidate agents on cells. In this embodiment, candidateagents (preferably libraries of candidate agents) are attached to beadson an array, preferably through a labile linker such as a photocleavablelinker. That is, candidate bioactive agents are synthesized on a bead(which may be porous), resulting in the candidate agents being attached,frequently via a scissile linkage, to the surface (which can be bothinternal and external) of a bead. The beads are distributed at discretesites on a surface as described herein for cells and microspheres, forexample a terminus of a fiber optic bundle, such that extremely largenumbers of assays can be simultaneously run. The candidate bioactiveagent is optionally cleaved from the bead, allowing free diffusionthrough the bead and interaction with the target cell. Detection ofbinding or activity is then accomplished in a variety of ways, as ismore fully outlined herein and known in the art.

Thus the present invention has a number of important advantages,including the use of very low amounts of reagents, a distinct spatiallocalization of the assays, decreased propensity to diffusion anddecreased rates of solvent evaporation. In addition, as is more fullyoutlined below, by varying the amount of candidate bioactive agentattached to each bead, the concentration of the candidate bioactiveagent can be varied, allowing the simultaneous determination ofconcentration effects. This is quite significant, as it allows thedetermination of kinetic parameters such as K_(m) and K_(i) and alsoavoids subsequent scale-up, including resynthesis of the agents andreassays.

The cells are contacted with the beads, either as a component of thebead (i.e. the cells are in or on the bead as outlined above) or bycontacting the surface of the array with a “lawn” of cells.

In a preferred embodiment, the cells and candidate agents are on beads.That is, the microspheres comprising the candidate agents aresynthesized, and then cells are associated with the beads; again, asoutlined above, this may be done in a variety of ways. For example,porous beads may be used and the cells either grown in the pores orentrapped in the pores. Similarly, when very porous substances such asagarose are used, the cells may be embedded in the beads. Alternatively,the cells may be associated with the external surface of the beads,similar to the manner that cells attach to planar surfaces. In thisembodiment, it may not be necessary to cleave the candidate agents offthe beads to see effects, particularly for agents that interact withcell surface moieties. However, cleavage of the agent off the bead canallow for diffusion into or onto a cell.

Alternatively, a preferred embodiment utilizes arrays comprising beadswith candidate agents which is later contacted with a substratecomprising cells. That is, an microsphere array is made (and decoded, ifnecessary, although decoding may not occur until after the assay) andthen contacted with cells, either as a “lawn” of cells that are grown ona substrate or tissue samples, etc. The bioactive agents are preferablycleaved off of the bead, for example using photolysis, and then diffuseeither onto or into the cells.

Suitable scissile linkages are described in U.S. Ser. No. 60/119,343,hereby incorporated by reference in its entirety.

The above and other features of the invention, including various noveldetails of construction and methods, and other advantages, will now bemore particularly described with reference to the accompanying claims.It will be understood to one skilled in the art that the particularapparatus and method embodying the invention are shown by way ofillustration and not as a limitation of the invention. The principlesand features of the invention may be employed in various and numerousembodiments without departing from the scope of the invention. Allreferences cited herein are expressly incorporated by reference in theirentirety.

1. A cellular array for detecting the response of individual cells to atleast one analyte of interest comprising: a) a substrate comprising aplurality of discrete sites; and b) a plurality of cells dispersed atsaid discrete sites, wherein each cell is encoded with at least oneexogenous binding partner.
 2. A cellular array according to claim 1,wherein said substrate is a fiber optic bundle.
 3. A cellular arrayaccording to claim 1 or 2, wherein said discrete sites comprise wells.4. A cellular array according to claim 1, 2 or 3 wherein said exogenousbinding partner is an antibody ligand.
 5. A cellular array according toclaim 1, 2, 3 or 4 wherein each cell is encoded with at least twoexogenous binding partners.
 6. A cellular array according to claim 1, 2,3, 4 or 5 wherein said exogenous binding partner is a fluorophorebinding peptide.
 7. A cellular array according to claim 1, 2, 3, 4, 5 or6 wherein said cells further comprise candidate agents.
 8. A method ofmaking a cellular array comprising: a) associating a population of cellswith a population of microspheres, such that each microsphere has atleast one associated cell, wherein said population comprises a firstsubpopulation comprising a first cell type and at least a secondsubpopulation comprising a second cell type, wherein said firstsubpopulation comprises at least a first exogenous binding partner andsaid second subpopulation comprises at least a second exogenous bindingpartner; b) distributing said population of microspheres onto discretesites of a substrate; and c) identifying the location of at least saidfirst and said second exogenous binding partners.
 9. A method accordingto claim 8 wherein said microspheres are porous and said cells areentrapped within said microspheres.
 10. A method according to claim 8wherein said cells are grown on the surface of said microspheres.
 11. Amethod according to claim 8 wherein said microspheres comprise a firstbinding ligand and said cells comprise a binding partner.
 12. A methodof screening comprising: a) contacting a candidate agent with a cellulararray comprising: i) a substrate comprising plurality of discrete sites;and ii) a plurality of cells dispersed at said discrete sites, whereineach cell is encoded with at least one exogeneous binding partner; andb) determining the effect of said candidate agent on said cells.
 13. Amethod of screening comprising: a) contacting cells with an arraycomprising: i) a substrate comprising a plurality of discrete sites; andii) a plurality of microspheres dispersed at said sites, wherein each ofsaid microspheres comprise a candidate bioactive agent.
 14. A methodaccording to claim 13 wherein said bioactive agents are linked to saidmicrospheres via a photolabile linkage.
 15. A method according to claim14 wherein said method further comprises releasing said candidatebioactive agents.